Diacylglycerol kinase (DGK) catalyzes the conversion of diacylglycerol (DG) to phosphatidic acid. DG is a key second messenger that regulates the activity of protein kinase C (PKC). PKC signaling pathways are of central importance for cell growth and differentiated function. Thus, one function of DGK is to attenuate DG signals, and consequently, PKC activity. We have cloned a unique cDNA with homology to known DGKs (pl40). The expressed partial cDNA has DGK catalytic activity. Proposed studies are designed to isolate the remaining coding region and to use structure-function studies with pl40 mutant constructs to study pl40 domain structure and organization. In other studies we have characterized agonist stimulated DG production and PKC isozyme activation in GH4C1 rat pituitary cells and normal and transformed REF52 fibroblasts. We will use these well-characterized cell lines to determine the effects of p140/DGK activity on agonist-stimulated DG production, PKC isozyme activation, PKC substrate phosphorylation and cellular responses. These studies will begin to define the role of p140 in regulating various DG pools. By correlating effects on DG pools with effects on PKCs, these studies will also help elucidate the relationship between various sources and pools of cellular DGs and PKC signaling events associated with cell growth, gene expression and transformation.